CN115088733A - Method for improving cold resistance of passion fruit based on planting Piriformospora indica - Google Patents

Method for improving cold resistance of passion fruit based on planting Piriformospora indica Download PDF

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CN115088733A
CN115088733A CN202210750878.1A CN202210750878A CN115088733A CN 115088733 A CN115088733 A CN 115088733A CN 202210750878 A CN202210750878 A CN 202210750878A CN 115088733 A CN115088733 A CN 115088733A
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passion fruit
indica
cold resistance
piriformospora
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陈发兴
李斌奇
施娟
梁晓毅
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Fujian Baiguo No1 Agricultural Development Co ltd
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract

The invention belongs to the technical field of plant production and cultivation, and discloses a method for improving cold resistance of passion fruit based on planting Piriformospora indica. And injecting the obtained bacterial liquid to the vicinity of the root of the passion fruit plant seedling, and injecting twice to obtain the passion fruit plant seedling implanted with endophytic fungus Pisum indicum at the root. The invention enhances the cold resistance of the passion fruit plants in low-temperature production areas in autumn and winter, improves the economic yield, and has important economic, social and ecological benefits for the sustainable development of the passion fruit industry.

Description

Method for improving cold resistance of passion fruit based on planting Piriformospora indica
Technical Field
The invention belongs to the technical field of plant production and cultivation, and particularly relates to a method for improving cold resistance of passion fruit based on planting Piriformospora indica.
Background
Passion fruit is a herbaceous vine of Passiflora in Passifloraceae, and is widely planted in Fujian, Guangxi, Taiwan, Yunnan and other places in China. Passion fruit has become an important economic industry in some areas of our country. Piriformospora indica is a root endophytic fungus found by Indian scientist Verma in the taler desert in northwest India in 1998, is an ideal endophytic fungus, has a wide host range, and has growth promoting and stress resisting effects on host plants.
Passion fruit is a temperature sensitive plant, and physiological disorders are easily caused by overhigh or lower temperature. In the process of passion fruit cultivation, fruits cannot be completely ripe due to low temperature in autumn and winter, and are easily affected by low temperature cold injury or freezing injury, the growth of seedlings is inhibited, leaves are wilted, plants grow slowly, even the growth is stopped, and great economic loss is caused to numerous fruit growers.
Disclosure of Invention
In order to improve the cold resistance of passion fruit seedlings, the invention aims to provide a method for improving the cold resistance of passion fruits based on planting Piriformis indica.
The application provides a method for improving cold resistance of passion fruit based on planting Piriformospora indica, which adopts the following technical scheme:
inoculating Piriformospora indica on a KM solid culture medium, and culturing in a dark constant temperature incubator at 28 ℃ for 5-6 days;
secondly, cutting mycelium blocks of the Piriformospora indica on the KM solid culture medium, and inoculating the mycelium blocks into a PDA liquid culture medium, wherein the PDA liquid culture medium is 200 g/L of potatoes, 20 g/L of glucose and 7 of PH value;
thirdly, placing the PDA liquid culture medium connected with the mycelium blocks in a shaking table at the temperature of 30 ℃ at 150 r/min for dark culture for 7 days;
(IV) crushing mycelium pellets in the bacterial liquid by using a juicer, and adding sterile water until the final concentration of the Piriformospora indica liquid is OD600 nm =1.121；
And (V) injecting the obtained Piriformospora indica liquid to the vicinity of the roots of passion fruit seedlings.
By adopting the technical scheme, the Indian pyricularia indica bacterial liquid is sprayed near the root of the passion fruit, so that the root system of the passion fruit is infected by the Indian pyricularia indica, and the cold resistance of the root system of the passion fruit is improved.
Preferably, step three further comprises: using a 250 mL conical flask, 200 mL of PDA broth was added to 2 pieces of mycelium per flask.
By adopting the technical scheme, 2 mycelium blocks are put into the PDA liquid culture medium, so that the propagation rate of the Pipriform indica can be increased on one hand, and the survival rate of the Pipriform indica can be increased on the other hand.
Preferably, the size of each mycelium block is about 1cm 2 。
By adopting the technical scheme, the hypha blocks are arranged to be 1cm 2 And the convenience of the inoculation operation process is improved.
Preferably, the root endophytic fungus pyricularia indica is injected twice near the root of the passion fruit seedling.
By adopting the technical scheme, the bonding efficiency of the Pistacia lentiscus and the roots of the passion fruit can be effectively improved by injecting the Pistacia lentinus seedlings at the roots twice, so that the infection is sufficient.
Preferably, the interval between two injections is 15 days.
By adopting the technical scheme, the injection time interval is set to be 15 days, so that the root system of the passion fruit can obtain sufficient adaptation time.
Preferably, the endophytic fungus Piriformospora indica in the root is injected near the root of the plant in an amount of 5 mL of bacterial solution per time.
By adopting the technical scheme, the bacterial liquid amount of 5 mL can meet the requirement of infiltrating the root systems of most passion fruits.
Preferably, the Piriformospora indica solution is injected at a location 5 cm from the plant stem.
By adopting the technical scheme, the bacterial liquid is injected at the position 5 cm away from the plant stem, so that the bacterial liquid can be contacted with the root ends of most of root systems, and the infection efficiency of the bacterial liquid and the root systems is improved.
Preferably, the Piriformospora indica solution is stored in a refrigerator at 4 ℃ for later use.
By adopting the technical scheme, the bacterial liquid of the Piriformospora indica can keep activity after being stored in a refrigerator at the temperature of 4 ℃.
Preferably, the passion fruit seedlings which are strong in growth are selected and have the height of 28-33 cm.
By adopting the technical scheme, the survival rate of the passion fruit plants can be improved by selecting the plants which grow robustly.
The invention has the advantages and positive effects that:
the invention discloses a method for enhancing cold resistance of passion fruit by implanting root endophytic fungus Piriformospora indica, which comprises the steps of selecting single seedlings of the passion fruit which grow vigorously as inoculated plants, preparing a PDA liquid culture medium, inoculating solid Piriformospora indica strains into the PDA liquid culture medium, and placing the PDA liquid culture medium in a shaking table for shake culture at room temperature. And injecting the obtained bacterial liquid to the vicinity of the root of the passion fruit plant seedling, and injecting twice to obtain the passion fruit plant seedling implanted with endophytic fungus Pisum indicum at the root.
Drawings
FIG. 1 is a schematic illustration of the effect of Cocculus indica on the low temperature phenotype of passion fruit;
FIG. 2 is a graph showing the effect of Piriformospora indica on the electrical conductivity and MDA content of passion fruit leaves subjected to low-temperature stress;
FIG. 3 is a graph showing the effect of Piriformospora indica on the low temperature stress of a passion fruit leaf osmoregulation substance;
FIG. 4 is a graph of the effect of Piriformospora indica on low temperature stress passion fruit leaf antioxidant enzymes;
FIG. 5 is a graph showing the effect of Piriformospora indica on the content of active oxygen in leaves of passion fruit subjected to low-temperature stress.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The present application is described in further detail below with reference to figures 1-5.
The application discloses a method for improving cold resistance of passion fruit based on planting Piriformospora indica.
(1) Inoculating Piriformospora indica on a KM solid culture medium, and culturing in a dark constant temperature incubator at 28 deg.C for 5 days, optionally 6 days;
formulation of KM solid Medium, 15.00 g agar, 2.00 g peptone, 1.00g yeast extract, 1.00g casein hydrolysate, 50mL 20X Salt stock solution, 10mL 100X Microelement stock solution, 10mL Fe-EDTA, 1mL 1000X Vitamin stock solution, H 2 0 930 mL；
Wherein 20X Salt stock solution: 12.00 g NaN0 3 、10.40 g KCl、10.40 g MgS04·7H 2 0、30.40 g KH 2 POa、1000 mL H 2 0；
100 X Microelement stock solution：2.20 g ZnSO 4 ·7H 2 0、1.00 g H 3 B0 3、 0.50 g MnS0 4 ·H 2 0、0.16 g CoC1·5H 2 0、0.16 g CuS04·5H 2 0、0.11 g (NHa) 6 MO 7 O 24 、1000 mL H 2 0；
Fe-EDTA：0.13 g FeSO 4 、0.18 g EDTA、100 mL H 2 0；
1000 x Vitamin stock solution：0.10 g Thiamin (B1)、0.04 g Glycin (H 2 NCH 2 COOH)、0.01 g Nicotinure (C 6 H 5 NO 2 )、0.01 g Pyridoxin (VitaminB6)、1000 mL H20；
Adjusting pH to 6.5-6.8 with 10% KOH, and sterilizing at 121 deg.C for 20 min.
(2) Preparing a PDA liquid culture medium: adding distilled water into 200 g potato, boiling to 1L, cooking potato into paste, filtering to remove potato, collecting supernatant, adding 20 g glucose, and neutralizing to pH 7. The prepared PDA liquid culture medium is subpackaged into 200 ml per bottle, and the bottle is a 250 ml conical bottle.
(3) Cutting mycelia of Pyricularia indica (L.) karst on KM solid culture medium, wherein each mycelia is about 1cm in size 2 Inoculating into the PDA liquid culture medium obtained in the step (2), inoculating 2 blocks into each bottle, and placing the PDA liquid culture medium inoculated with the mycelium blocks in a shaking table at 150 r/min and 30 ℃ for dark culture for 7 days.
(4) Extracting the bacterial liquid obtained in step (3) by using a syringe to obtain 20ml, and squeezing juicePulverizing mycelium group in bacteria liquid, adding sterile water until final concentration of bacteria liquid is OD600 nm =1.121。
(5) Selecting healthy individual seedlings of 'Fujian passion fruit No. 1' variety passion fruit as inoculated plants, wherein the height of the plants is 28-33 cm, and the concentration is OD600 nm Injecting bacterial liquid of =1.121 to the vicinity of the root of the passion fruit seedling, wherein the injection amount is 5 ml each time, the injection position is 5 cm away from the stem of the passion fruit seedling, and the endophytic fungus Pilus indica at the root is injected twice in the vicinity of the root of the passion fruit seedling, and the interval time between the two injections is 15 days. Obtaining the seedlings of the passion fruit which are inoculated with the endophytic fungus Piriformospora indica of roots.
1 materials and methods
To test the effectiveness of the method, the seedlings of passion fruit were randomly divided into 20 strains each of the control group (CK) and the Piriformospora indica treatment group. The Piriformospora indica treatment group was treated in the above manner, and the control group was distilled water. And then culturing the seedlings at 25 ℃ for 7 days, carrying out low-temperature stress treatment at 0 ℃ for 0, 6, 12 and 24 hours, recording the phenotype characteristics of the plants subjected to low-temperature stress, taking 2-3 true leaves at the top for measuring physiological and biochemical indexes, and setting 5 biological repeats for each treatment.
1.1 measurement of physiological and biochemical indices
Identifying the root system symbiotic condition of the Piriformospora indica and the passion fruit by adopting a trypan blue staining method; measuring the relative conductivity of the passion fruit leaves by a reference conductivity method; methods such as royal jelly and the like for measuring the soluble sugar content, the soluble protein content, the proline (Pro) content, the Ascorbate Peroxidase (APX) activity, the superoxide dismutase (SOD) activity, the Catalase (CAT) and the Malondialdehyde (MDA) concentration; measuring Glutathione Reductase (GR) content, Glutathione (GSH) content, polyphenol oxidase (PPO) activity, and Hydrogen peroxide (H) 2 O 2 ) Concentration and Superoxide anion (O2) - ) Concentration reference Caokang, etc.
2 results and analysis
2.1 symbiosis of Piriformospora indica with Passion fruit
Referring to FIG. 1-A, PDA liquid medium (content: content)P. indicaMycelium blocks) are placed on a shaking table for shaking culture, the mycelium grows in a block shape, and the mycelium gradually becomes larger along with the increase of the block shape. With reference to FIG. 1-B, inoculationP. indica After 15 days, detecting the permanent planting condition of the root system of the passion fruit, and clearly observing round or pear-shaped chlamydospores of Piriformis indica on the epidermis of the root of the passion fruit under a 10-fold microscope, which shows thatP. indicaSuccessfully planted in the root system of the passion fruit.
2.2 Effect of Piriformospora indica treatment on Passion fruit seedling phenotype
Referring to fig. 1-C, at the beginning of low temperature stress, there was no significant difference in the phenotype of passion fruit in both groups of treatments, whereas the low temperature damage symptoms were significantly different in both groups after 24 h treatment at 0 ℃. The CK group seedling leaves show frozen symptoms such as curling, wilting and water stain;P. indicacold injury symptoms also appeared in the group, but the leaf curl was significantly less than in the CK group, and the water stain area was less.
2.3 Effect of Piriformospora indica on cell membranes of passion fruit leaves under Low temperature stress
Referring to fig. 2-a, passion fruit leaf conductivity increases with time to low temperature stress. After 12 h and 24 h of low-temperature stress, the relative conductivity of the passion fruit leaves planted with Piriformospora indica is remarkably lower than that of a control group (P is less than 0.05), and is respectively reduced by 21.51% and 24.30%.
Referring to FIG. 2-B, the MDA content of the passion fruit leaves increases after low temperature stress. After the low-temperature stress for 12 h and 24 h, the content of the control group MDA is increased by 2.28 times and 2.74 times of that of the control group MDA after 0 h; it is worth noting thatP. indicaThe treated passion fruit leaves are significantly lower than those withoutP. indicaTreated plants (P < 0.05), reduced by 25.86% and 18.65%, respectively.
2.4 Effect of Piriformospora indica on Pogostemon osmoregulation substances under Low temperature stress
Referring to fig. 3-a, the proline Pro content tends to increase with the increase of the low-temperature treatment time. The Pro content difference between the two groups of treatments was not significant after 0 h and 6 h (P)>0.05); when the stress is carried out for 12 h and 24 h at low temperature,the proline Pro content is obviously different from that of a control group (P is less than 0.05),P. indicathe proline Pro content of the treated passion fruit leaves is enhanced by 35.72 percent and 36.42 percent;p. indica treatment reached significant levels throughout (a)P < 0.05), 108.18% increase in 24 h compared with 0 h.
Referring to FIG. 3-B, during the initial stage of low temperature stress, the soluble protein content of both treatments gradually increased.P. indicaNo significant difference 6 h before treatment (P)>0.05), 12 h and 24 h were significantly higher than the control group and 20.79% and 38.51%.
Referring to FIG. 3-C, soluble sugars show an increasing trend with the extension of low temperature stress time.P. indicaThe content of soluble sugar in the passion fruit leaves is obviously higher than that in the CK group (P is less than 0.05) after low-temperature stress is carried out for 6 hours, 12 hours and 24 hours; the soluble sugar content after 24 h was 1.3 times that of the control group.
2.5 Effect of Piriformospora indica on Passion fruit antioxidant enzymes under Low temperature stress
Referring to FIG. 4-ABC, under low temperature stressP. indicaThe treatment enhanced the SOD activity, APX activity and CAT activity of the passion fruit leaves. Referring to FIG. 4-A, the channelP. indicaAnd (4) treating, wherein the SOD enzyme activity of the passion fruit leaves shows an ascending trend at low temperature. The control group shows an ascending trend in the initial stage of low-temperature stress, and the SOD starts to decline after reaching the peak value of 604.32U/g after reaching 12 hours; compared with the contrast group, the ratio of the composition,P. indicathe 6 h and 12 h of the treatment are significantly higher than the control group (P < 0.05); referring to fig. 4-B, the APX activity shows an increasing trend with the extension of low temperature stress time, and the APX activity of the passion fruit plant leaves inoculated with pyricularia indica is significantly higher than that of the control group (P < 0.05), and is increased by 23.79%, 28.27% and 32.21% at 6 h, 12 h and 24 h respectively; with reference to figure 4-C of the drawings,P. indicathe treated CAT activity showed a significant trend of rising, the control group showed a trend of rising-falling-rising, and the CAT activity showed a trend of rising-falling-rising when the stress was 12 h and 24 hP. indicaThe CAT activity of the treated group is obviously higher than that of the control group, and is respectively increased by 60.16% and 49.10%.
Referring to FIG. 4-DEF, under low temperature stressP. indicaTreatment for inhibiting PPO activity and H of passion fruit leaves 2 O 2 Concentration sum O2 - The increase in concentration. The PPO activity of the control group shows a trend of increasing first and then decreasing, and the PPO is induced within 12 h of stressThe activity rose rapidly (0.66U/g) and then began to decline; whileP. indicaThe PPO activity of the treatment slowly increased within 24 h and was significantly lower than that of the control group (P < 0.05), and after 6 h and 12 h of low-temperature stress, the PPO activity was reduced by 63.49% and 81.06%, respectively. Two groups of H 2 O 2 The concentration shows the trend of rising firstly and then falling, the control group rapidly rises after the low temperature stress is carried out for 6 hours, the concentration is increased by 43.55 percent compared with 0 hour, and the concentration is gradually reduced along with the extension of the stress time; and thenP. indicaThe trend of the treatment groups was more gradual, increasing slowly and then decreasing slowly.P. indicaH of treatment group 2 O 2 The content of the strain is obviously different from that of a control group at 6 h, 12 h and 24 h under stress (P < 0.05), and is respectively reduced by 28.49%, 9.96% and 13.63%. Two sets of O2 - The concentration is dynamically changed in the whole low-temperature stress processP. indicaGroups were significantly lower than the control group.
2.6 Effect of Piriformospora indica on antioxidant Activity of Passion fruit leaves under Low temperature stress
Referring to fig. 5-a, the GSH activity of the passion fruit leaves increases gradually overall as the time to cold stress increases. When the temperature is lower than the control group, the treatment time is 0 hP. indicaThe difference of the GSH activity of the group is not obvious, the GSH activity is increased by 28.69 percent when the stress is carried out for 6 hours, and the GSH activity is the highest (2.26 mu mol/g) when the low-temperature stress is up to 12 hours and is increased by 23.23 percent.
Referring to FIG. 5-B, control group was subjected to low temperature stressP. indicaThe GR activity of the group shows a trend of increasing first and then decreasing with the increase of the low-temperature stress time, and the GR activity of the two groups reaches a peak value (162.71 and 197.38 nmol/min/g) at 12 h and then begins to decrease. When the temperature is low for 0 h, the GR activity is not significantly different from that of a control group; when the stress is carried out for 6 h and 12 h at low temperature,P. indicathe GR activity of the treated group is obviously higher than that of the control group (P < 0.05), and is respectively improved by 14.71 percent and 21.31 percent, and is reduced by 24 hours of stress.
In conclusion, after the Piriformospora indica is colonized, the antioxidant enzyme activity of the passion fruit leaves is obviously improved, and the capability of eliminating Reactive Oxygen Species (ROS) is improved. The Indian pyricularia indica symbiotic plant has stable cell membrane and strong oxidation resistance, and improves the cold resistance of the seedlings.
The above embodiments are preferred embodiments of the present application, and the protection scope of the present application is not limited by the above embodiments, so: equivalent changes in structure, shape and principle of the present application shall be covered by the protection scope of the present application.

Claims (9)

1. A method for improving cold resistance of passion fruit based on planting Piriformospora indica is characterized in that:
inoculating Piriformospora indica on a KM solid culture medium, and culturing in a dark constant temperature incubator at 28 ℃ for 5-6 days;
secondly, cutting mycelium blocks of the Piriformospora indica on the KM solid culture medium, and inoculating the mycelium blocks into a PDA liquid culture medium, wherein the PDA liquid culture medium is 200 g/L of potatoes, 20 g/L of glucose and 7 of PH value;
thirdly, placing the PDA liquid culture medium connected with the mycelium blocks in a shaking table at the temperature of 30 ℃ at 150 r/min for dark culture for 7 days;
(IV) crushing mycelium pellets in the bacterial liquid by using a juicer, and adding sterile water until the final concentration of the Piriform solonchaete liquid is OD600 nm =1.121；
And (V) injecting the obtained Piriformospora indica liquid to the vicinity of the roots of passion fruit seedlings.
2. The method for improving cold resistance of passion fruit based on the implantation of Piriformospora indica according to claim 1, wherein the method comprises the following steps: the third step also comprises: using a 250 mL conical flask, 200 mL of PDA broth was added to 2 pieces of mycelium per flask.
3. The method for improving the cold resistance of passion fruit based on the implantation of pyricularia indica according to claim 1, wherein the method comprises the following steps: the size of each mycelium block is about 1cm 2 。
4. The method for improving the cold resistance of passion fruit based on the implantation of pyricularia indica according to claim 1, wherein the method comprises the following steps: the endophytic fungus Piriformospora indica from root is injected twice near the root of the seedling of passion fruit.
5. The method for improving the cold resistance of passion fruit based on the implantation of pyricularia indica according to claim 4, wherein the method comprises the following steps: the interval between the two injections was 15 days.
6. The method for improving the cold resistance of passion fruit based on the implantation of pyricularia indica according to claim 4, wherein the method comprises the following steps: the root endophytic fungus Piriformospora indica has 5 mL of bacterial liquid per injection near the root of the plant.
7. The method for improving cold resistance of passion fruit based on the implantation of Piriformospora indica according to claim 4, wherein the method comprises the following steps: the Piriformospora indica solution was injected 5 cm from the plant stem.
8. The method for improving the cold resistance of passion fruit based on the implantation of pyricularia indica according to claim 1, wherein the method comprises the following steps: the Piriformospora indica liquid is stored in a refrigerator at 4 ℃ for later use.
9. The method for improving cold resistance of passion fruit based on the implantation of Piriformospora indica according to claim 1, wherein the method comprises the following steps: selecting strong passion fruit seedlings with the height of 28-33 cm.
CN202210750878.1A 2022-06-29 2022-06-29 Method for improving cold resistance of passion fruit based on planting Piriformospora indica Pending CN115088733A (en)

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CN105830681A (en) * 2016-03-21 2016-08-10 重庆步步康农业发展有限公司 Overwintering planting method for passion fruits
CN113151008A (en) * 2021-04-19 2021-07-23 浙江大学 Method for improving nutrient utilization efficiency of watermelons by utilizing endophytic fungi

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Publication number Priority date Publication date Assignee Title
WO2013110258A1 (en) * 2012-01-24 2013-08-01 Hochschule Anhalt Antifungal formulations for combatting plant diseases
CN105830681A (en) * 2016-03-21 2016-08-10 重庆步步康农业发展有限公司 Overwintering planting method for passion fruits
CN113151008A (en) * 2021-04-19 2021-07-23 浙江大学 Method for improving nutrient utilization efficiency of watermelons by utilizing endophytic fungi

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